INTRODUCTION

Cancer is one of the major causes of death worldwide. The development of identification and treatment are important for cancer treatment. However, effective and selective treatment methods are insufficient against some types of cancer. Scientists continue their studies to find effective molecules for cancer treatment.^1,2 Compounds bearing nitrogen, sulfur, and oxygen play a significant role by forming hydrogen bonds with DNA.³ Therefore heterocyclic compounds such as pyridine and pyrimidine showed anticancer,⁴ antibacterial,⁵ antifungal,⁶ analgesic, and anti-inflammatory activity.⁷

Sorafenib, which carries a pyridine ring and urea group was confirmed by the United States Food and Drug Administration for the treatment of renal cell carcinoma in 2005. Sorafenib, which has a broad spectrum for anticancer therapy, inhibits some kinases such as vascular endothelial and platelet-derived growth factor. Therefore, sorafenib could be used with numerous types of cancer. Chemical structures of compounds were envisioned consisting of three parts: pyridyl moiety, a linker, and urea functional group as a pharmacophore (Scheme 1).⁸

Wang et al.⁹ studied new benzimidazole-2-urea derivatives that decrease the proliferation of some cancer cells (HeLa, K562, HepG2) and reported that these molecules could be used as tubulin inhibitors. In another study, Fortin et al.¹⁰ discovered that compounds containing urea derivatives had greater antiproliferative activity than amide groups from their structure activity relationship. De et al.¹¹ synthesized N’-(2-(4-substitute)cyclopropanecarbonyl)isonicotinohydrazide-containing carbohydrazide and a pyridine ring and evaluated their cytotoxic activity against A549, PC3, and U373 cells.

Pyridine is an important ring system with numerous biologic activities. For example, Kurumurthy et al.¹² selected pyridine derivatives to achieve cytotoxic activity against THP1, U937, HL60 and B16-F10 cells.

Using results obtained in the literature and pharmacophore analysis (Scheme 1), target molecules carrying urea and carbohydrazide derivatives were synthesized from methyl 6-aminopyridine-3-carboxylate and evaluated their cytotoxic activity against cancer cells (HCC1937, Capan1, MCF-7, HeLa and MRC5).

MATERIALS AND METHODS

Chemistry

All chemicals reagents and solvents were obtained from Sigma-Aldrich (St. Louis, MO, USA), and Merck (Darmstadt, Germany). The homogeneity and purity of the compounds were checked using thin-layer chromatography (TLC), performed on commercially available silica gel (Kieselgel 60, F254) coated aluminum sheets (Merck) by using petroleum ether:ethyl acetate (10:90 v/v) as the solvent system. Visualization on TLC was performed using ultraviolet light (λ=254 nm) and an iodine indicator. Melting points (MP) were determined using a Schmelzpunktbestimmer SMP II. IR spectra were recorded with a Shimadzu FTIR-8400S (Japan). ¹H-NMR spectra were recorded on a Bruker Avance 400 MHz (USA) in DMSO-d₆ using tetramethylsilane (TMS) as the internal reference. Chemical shifts (δ) were expressed in parts per million relative to TMS and the following abbreviations were used to describe the peak patterns when appropriate: s, (singlet); d, (doublet); t, (triplet); m, (multiplet). Elemental analysis (C, H and N) was performed on a CHNS-Thermo Scientific Flash 2000 (Waltham, MA USA).

Synthesis of urea derivatives

Methyl 6-aminopyridine-3-carboxylate was dissolved in acetone at 80°C. Then, a solution of the corresponding equimolar isocyanate in dry acetone was added as two parts, every 30 minutes. The reaction mixture was refluxed for 6 hours. The reaction was finalized by checking with TLC and left overnight. The precipitate was filtered off, dried, and purified with acetone.¹³

Methyl 6-(3-(4-nitrophenyl)ureido)nicotinate (1)

Yellow solid; Yield: 70%; MP: 237-239°C; IR (v_max cm^-1): 3365, 3211 (N-H), 3080 (=C-H stretching), 2983, 2843 (C-H), 1708 (urea C=O), 1604, 1562, 1506, 1491, 1411 (C=C, NO₂, N-H bending, C-N), 1273 (C-O), 839 (=C-H). ¹H-NMR (DMSO-d₆/TMS, 400 MHz, δ in ppm): 3.83 (s, 3H, -OCH₃), 7.69-8.84 (m, 7H, Ar-H), 9.99 (s, 1H, NH), 10.73 (s, 1H, NH). For C₁₄H₁₂N₄O₅ (M.W.: 316.27 g/mol) calculated (%): C:53.17, H:3.82, N:17.71. Found: C:54.46, H:3.66, N:17.95.

Synthesis of the hydrazide derivatives

Methyl 6-(3-(4-nitrophenyl)ureido)nicotinate was dissolved in ethanol on a magnetic stirrer. Then, hydrazine monohydrate (1 mL) was added. The reaction mixture was refluxed for 6 hours. The mixture was filtered and washed with methanol.¹⁴

1-(5-(Hydrazinecarbonyl)pyridin-2-yl)-3-(4-nitrophenyl)urea (2)

Yellow solid; Yield: 65%; MP: 274-275°C; IR (v_max cm^-1): 3338, 3221 (N-H), 3078 (=C-H stretching), 1712 (C=O urea), 1678 (C=O hydrazide), 1620 (C=N), 1562, 1510, 1491, 1431 (C=C, NO₂, N-H, C-N), 815 (=C-H). ¹H-NMR (DMSO-d₆/TMS, 400 MHz, δ in ppm): 4.48 (s, 2H, NH₂), 7.71-8.87 (m, 7H, Ar-H), 9.93 (s, 1H, NH), 10.04 (s, 1H, NH), 10.78 (s, 1H, NH). C₁₃H₁₂N₆O₄ (M.W.: 316.27 g/mol) calculated (%): C:49.37, H:3.82, N:26.57. Found: C:51.05, H:3.97, N:25.25.

Synthesis of the carbohydrazide derivatives

To a solution of hydrazide (1 mmol) (2) and triethylamine (2 mmol) in dry CH₂Cl₂ (5 mL), a solution of previously prepared benzoyl chloride (1 mmol) was added dropwise at room temperature. The reaction mixture was stirred on a magnetic stirrer for 3 hours. The precipitate was then washed with distilled water and filtered. The purity of compounds was checked with TLC.¹⁵

1-(5-(2-(4-Fluorobenzoyl)hydrazinecarbonyl)pyridin-2-yl)-3-(4-nitrophenyl)urea (3a)

Yellow solid; Yield: 70%; MP: 257-259°C; IR (v_max cm^-1): 3304, 3207, 3122 (N-H), 3080 (=C-H stretching), 1712 (C=O), 1612, 1562, 1508, 1492 (C=C, NO₂, N-H, C-N), 842 (=C-H). ¹H-NMR (DMSO-d₆/TMS, 400 MHz, δ in ppm): 7.67-8.84 (m, 11H, Ar-H), 9.69 (s, 1H, NH), 10.04 (s, 1H, NH), 10.84 (s, 2H, NH). C₂₀H₁₅FN₆O₅ (M.W.: 438.37 g/mol) calculated (%): C:54.80, H:3.45, N:19.17. Found: C:55.24, H:3.22, N:18.65.

1-(5-(2-(4-Chlorobenzoyl)hydrazinecarbonyl)pyridin-2-yl)-3-(4-nitrophenyl)urea (3b)

Yellow solid; Yield: 75%; MP: 202-203°C; IR (v_max cm^-1): 3304, 3207, 3122 (N-H), 3080 (=C-H stretching), 1712 (C=O), 1612, 1562, 1508, 1492, 1431 (C=C, NO₂, N-H, C-N), 842 (=C-H). ¹H-NMR (DMSO-d₆/TMS, 400 MHz, δ in ppm): 7.71-8.85 (m, 11H, Ar-H), 9.98 (s, 2H, NH), 10.73 (s, 2H, NH). C₂₀H₁₅ClN₆O₅ (M.W.: 454.82 g/mol) calculated (%): C:52.81, H:3.32, N:18.48. Found: C:52.75, H:3.48, N:18.44.

1-(5-(2-(4-Nitrobenzoyl)hydrazinecarbonyl)pyridin-2-yl)-3-(4-nitrophenyl)urea (3c)

Yellow solid; Yield: 80%; MP: 273-275°C; IR (v_max cm^-1): 3215, 3124 (N-H), 3082 (=C-H stretching), 1714 (C=O), 1612, 1564, 1510, 1492 (C=C, NO₂, N-H, C-N), 846 (=C-H). ¹H-NMR (DMSO-d₆/TMS, 400 MHz, δ in ppm): 7.68-8.85 (m, 11H, Ar-H), 9.62 (s, 1H, NH), 10.00 (s, 1H, NH), 10.74 (s, 2H, NH). C₂₀H₁₅N₇O₇ (M.W.: 465.38 g/mol) calculated (%): C:51.62, H:3.25, N:21.07. Found: C:52.33, H:3.12, N:20.66.

1-(5-(2-(2,6-Dichlorobenzoyl)hydrazinecarbonyl)pyridin-2-yl)-3-(4-nitrophenyl)urea (3d)

Yellow solid; Yield: 70%; MP: 258-260°C; IR (v_max cm^-1): 3369, 3215, 3122 (N-H), 3084 (=C-H), 1712 (C=O), 1612, 1566, 1492, 1481 (C=C, NO₂, N-H, C-N), 1031 (C-Cl), 844 (=C-H). ¹H-NMR (DMSO-d₆/TMS, 400 MHz, δ in ppm): 7.78-8.68 (m, 10H, Ar-H), 9.43 (s, 1H, NH), 10.00 (s, 1H, NH), 10.75 (s, 2H, NH). C₂₀H₁₄Cl₂N₆O₅ (M.W.: 489.27 g/mol) calculated (%): C:49.10, H:2.88, N:17.18. Found: C:50.35, H:2.97, N:16.85.

1-(5-(2-(4-Methylbenzoyl)hydrazinecarbonyl)pyridin-2-yl)-3-(4-nitrophenyl)urea (3e)

Yellow solid; Yield: 65%; MP: 275-277°C; IR (v_max cm^-1): 3369, 3205, 3122 (N-H), 3049 (=C-H stretching), 2987 (C-H), 1712 (C=O), 1610, 1562, 1508, 1492, 1431 (C=C, NO₂, N-H, C-N), 844 (=C-H). ¹H-NMR (DMSO-d₆/TMS, 400 MHz, δ in ppm): 2.39 (s, 3H, -CH₃), 7.47-8.81 (m, 11H, Ar-H), 9.45 (s, 1H, NH), 10.06 (s, 1H, NH), 10.77 (s, 2H, NH). C₂₁H₁₈N₆O₅ (M.W.: 434.41 g/mol) calculated (%): C:58.06, H:4.98, N:19.15. Found: C:57.23, H:5.05, N:18.56.

1-(5-(2-(4-Bromobenzoyl)hydrazinecarbonyl)pyridin-2-yl)-3-(4-nitrophenyl)urea (3f)

Yellow solid; Yield: 75%; MP: 240-241°C; IR (v_max cm^-1): 3207, 3122 (N-H), 3082 (=C-H stretching), 1712 (C=O), 1610, 1562, 1508, 1491, 1431 (C=C, NO₂, N-H, C-N), 844 (=C-H). ¹H-NMR (DMSO-d₆/TMS, 400 MHz, δ in ppm): 7.45-8.81 (m, 11H, Ar-H), 9.36 (s, 1H, NH), 10.20 (s, 1H, NH), 10.84 (s, 2H, NH). C₂₀H₁₅BrN₆O₅ (M.W.: 499.27 g/mol) calculated (%): C:48.11, H:3.03, N:16.83 Found: C:47.48, H:3.25, N:16.92.

1-(4-nitrophenyl)-3-(5-(2-(4-(trifluoromethyl)benzoyl)hydrazinecarbonyl)pyridin-2-yl)urea (3g)

Yellow solid; Yield: 70%; MP: 299-300°C; IR (v_max cm^-1): 3333, 3271, 3213 (N-H), 3080 (=C-H stretching), 1712 (C=O), 1680 (C=O hydrazide), 1614 (C=N), 1564, 1489, 1431, 1411 (C=C, NO₂, N-H, C-N), 1261 (C-F), 890 (=C-H). ¹H-NMR (DMSO-d₆/TMS, 400 MHz, δ in ppm): 7.64-8.84 (m, 11H, Ar-H), 9.75 (s, 1H, NH), 9.97 (s, 1H, NH), 10.72 (s, 2H, NH). C₂₁H₁₅F₃N₆O₅ (M.W.: 488.38 g/mol) calculated (%): C:51.65, H:3.10, N:17.21 Found: C:51.48, H:3.25, N:16.92.

1-(5-(2-(4-methoxybenzoyl)hydrazinecarbonyl)pyridin-2-yl)-3-(4-nitrophenyl)urea (3h)

Yellow solid; Yield: 75%; MP: 222-223°C; IR (v_max cm^-1): 3275, 3171, 3117 (N-H), 3080 (=C-H stretching), 2978 (C-H asymmetric stretching), 2841 (C-H symmetric stretching), 1703 (C=O), 1662 (C=O hydrazide), 1633 (C=N), 1537, 1506, 1499, 1471 (C=C, NO₂, N-H, C-N), 1327 (C-O), 824 (=C-H). ¹H-NMR (DMSO-d₆/TMS, 400 MHz, δ in ppm): 3.84 (s, 3H, OCH₃), 7.05-8.84 (m, 11H, Ar-H), 9.79 (2s, 1H, NH), 10.45 (2s, 1H, NH), 10.72 (s, 2H, NH). C₂₁H₁₈N₆O₆ (M.W.: 450.40 g/mol) calculated (%): C:56.00, H:4.03, N:18.66 Found: C:56.48, H:3.25, N:18.92.

1-(5-(2-benzoylhydrazinecarbonyl)pyridin-2-yl)-3-(4-nitrophenyl)urea (3i)

Yellow solid; Yield: 70%; MP: 203-204°C; IR (v_max cm^-1): 3271, 3213, 3124 (N-H), 3045 (=C-H stretching), 1712 (C=O), 1666 (C=O hydrazide), 1641 (C=N), 1602, 1562, 1506, 1489, 1431 (C=C, NO₂, N-H, C-N), 846 (=C-H). ¹H-NMR (DMSO-d₆/TMS, 400 MHz, δ in ppm): 7.45-8.84 (m, 11H, Ar-H), 9.80 (2s, 1H, NH), 10.68 (2s, 1H, NH), 11.48 (s, 2H, NH). C₂₀H₁₆N₆O₅ (M.W.: 420.38 g/mol) calculated (%): C:57,14 H:3.84, N:19.99 Found: C:57.48, H:3.65, N:19.92.

1-(4-nitrophenyl)-3-(5-(2-(4-(trifluoromethylthio)benzoyl)hydrazinecarbonyl)pyridin-2-yl)urea (3j)

Yellow solid; Yield: 65%; MP: 277-278°C; IR (v_max cm^-1): 3333, 3221 (N-H), 3080 (=C-H stretching), 1714 (C=O urea), 1680 (C=O hydrazide), 1641 (C=N), 1564, 1510, 1492 (C=C, NO₂, N-H, C-N), 844 (=C-H). ¹H-NMR (DMSO-d₆/TMS, 400 MHz, δ in ppm): 7.63-8.83 (m, 11H, Ar-H), 9.79 (2s, 1H, NH), 9.97 (s, 1H, NH), 10.72 (s, 2H, NH). C₂₁H₁₅F₃N₆O₅S (M.W.: 520.44 g/mol) calculated (%): C:48.46, H:2.91, N:16.15 Found: C:48.76, H:3.05, N:16.92.

Biology

Cell culture

A human pancreatic adenocarcinoma Capan1 cell line, human cervix carcinoma HeLa cell line, human lung fibroblasts MRC5 cell line, human breast adenocarcinoma MCF7 and HCC1937 cell line were obtained from American Type Culture Collection (Bethesda), and maintained as exponentially growing monolayers by culturing according to the supplier’s instructions in a humidifier incubator at 37°C supplied with 5% CO₂. All cell culture reagents were purchased from Biological Industries (Israel).

Cytotoxicity test

The cytotoxic potencies of the test compounds were determined using a WST1 Cell Protliferation Assay (Roche) according to the manufacturer’s instructions. All tested compounds were dissolved in DMSO. Cells were seeded into a 96-well plate at a density of 5000 cells/well for HeLa, MCF7 and HCC1937, 7500 cells/well for Capan1 and 10,000 cells per well for MRC5. The next day, cells were treated with compounds with the final concentrations of 1, 2, 5, 10, 25 µM and incubated for 48 hours at conventional cell culture conditions. DMSO was used a negative solvent control, and doxorubicin was included in the study as a positive cytotoxic control compound. The ratio of surviving cells after compound treatment was determined using a colorimetric WST-1 assay (Roche) as indicated in the protocol provided by manufacturer. The absorbance was measured using a Varioscan microplate reader (Thermo) at 450 nm with a 620-nm reference filter. To determine the IC₅₀ values, a sigmoid-dose response curve was fitted to the data using nonlinear regression on GraphPad Prism 5 software.

This study did not need ethics committee approval because in vitro methods were used for biologic activity processes.

RESULTS AND DISCUSSION

The synthetic route to the target compounds is outlined in Scheme 2. The structures of the compounds (1, 2, 3a-j) were confirmed using IR, ¹H-NMR, and elemental analysis. IR spectra of the compounds (1, 2, 3a-j) afforded N-H stretching (3115-3369) bands. IR spectra of all compounds (1, 2, 3a-j) were described C-H stretching (3045-3082), urea and carbohydrazide C=O stretching (1662-1714) bands, aromatic rings C=C stretching and NO₂ stretching (1411-1604) bands. The NH protons of carbohydrazide and urea groups resonated as two different singlet peak at 9.36-11.48 ppm. The aromatic protons displayed a multiplet at 7.05-8.85 ppm. The elemental analysis of compounds was in agreement with the proposed structures of the compounds.

It is known that some PARP inhibitors are highly-selective promising agents against cancer cells with homologous recombination (HR) DNA repair pathway deficiencies such as those harboring mutations on tumor suppressors BRCA1 or BRCA2 via generation of chromatid breaks, cell cycle arrest and apoptosis.^16,17 Therefore, we included HCC1937 and Capan1, which are defective in BRCA1 and BRCA2, respectively. We aimed to compare the cytotoxicity of the compounds on these cell lines with their activities on HR-proficient cancer cell lines (HeLa and MCF7) and also a non-tumoral MRC5 fibroblast cell line.

When the substitution pattern at the phenyl ring was determined, the effect of electron donor and electron acceptor groups on activity was considered. Therefore, methyl, methoxy, and halogens such as F, Cl, Br were selected as electron donors and a nitro group was selected as an electron acceptor. Our data suggest that only 3a having fluoro, 3b having chloro, 3c having nitro and 3d having 2,6 dichloro substituents had cytotoxic activities at the tested concentrations. The IC₅₀ values of these compounds are given in Table 1. Compounds 3e-j showed less cytotoxic activity on all cancer cells compared with 3a-d. However, our results suggest that the compounds possessed no selectivity toward HR defective Capan1 and HCC1937 cells harboring BRCA mutations compared with MCF7 and HeLa cells with intact HR pathways.

CONCLUSIONS

In the present paper, we reported the synthesis of some new urea and carbohydrazide derivatives from methyl 6-aminopyridine-3-carboxylate. The synthesized compounds were evaluated for their cytotoxic activity. Our data indicate that 3a-d are more selective to cancer cells compared with nontumoral fibroblasts; however, these compounds are not more potent on HR defective cells with BRCA mutants.

ACKNOWLEDGEMENTS

This study was supported by TÜBİTAK 215S112 and Marmara University Scientific Research Commission (project number: SAG-C-DRP-100616-0260).

Conflict of Interest: No conflict of interest was declared by the authors.